Method for identifying cells

ABSTRACT

Disclosed are silica-coated nanoparticles and a process for producing silica-coated nanoparticles. Silica-coated nanoparticles are prepared by precipitating nano-sized cores from reagents dissolved in the aqueous compartment of a water-in-oil microemulsion. A reactive silicate is added to coat the cores with silica. Also disclosed are methods for functionalizing silica-coated nanoparticles for use in a variety of applications.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patentapplication Ser. No. 09/572,469 filed May 17, 2000, entitled “CoatedNanoparticles.”

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with U.S. government support under grantnumber N00014-98-1-0621 awarded by the Office of Naval Research andgrant number NSF BIO-9871880 awarded by the National Science Foundation.The U.S. government may have certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the field of nanoparticles andmethods of making nanoparticles. More particularly, the inventionrelates to silica-coated nanoparticles prepared by using microemulsions.

BACKGROUND OF THE INVENTION

[0004] Nanoparticles are very small particles typically ranging in sizefrom as small as one nanometer to as large as several hundred nanometersin diameter. Their small size allows nanoparticles to be exploited toproduce a variety of products such as dyes and pigments; aesthetic orfunctional coatings; tools for biological discovery, medical imaging,and therapeutics; magnetic recording media; quantum dots; and evenuniform and nanosize semiconductors.

[0005] Nanoparticles can be simple aggregations of molecules or they canbe structured into two or more layers of different substances. Forexample, simple nanoparticles consisting of magnetite or maghemite canbe used in magnetic applications (e.g., MRI contrast agents, cellseparation tools, or data storage). See, e.g., Scientific and ClinicalApplications of Magnetic Microspheres, U. Häfeli, W. Schütt, J. Teller,and M. Zborowski (eds.) Plenum Press, New York, 1997; Sjøgren et al.,Magn.Reson. Med. 31: 268, 1994; and Tiefenauer et al., BioconjugateChem. 4:347, 1993. More complex nanoparticles can consist of a core madeof one substance and a shell made of another.

[0006] Many different type of small particles (nanoparticles ormicron-sized particles) are commercially available from severaldifferent manufacturers including: Bangs Laboratories (Fishers, Ind.);Promega (Madison, Wis.); Dynal Inc.(Lake Success, N.Y.); AdvancedMagnetics Inc.(Surrey, U.K.); CPG Inc.(Lincoln Park, N.J.); CortexBiochem (San Leandro, Calif.); European Institute of Science (Lund,Sweden); Ferrofluidics Corp. (Nashua, N.H.); FeRx Inc.; (San Diego,Calif.); Immunicon Corp.; (Huntingdon Valley, Pa.); MagneticallyDelivered Therapeutics Inc. (San Diego, Calif.); Miltenyi Biotec GmbH(USA); Microcaps GmbH (Rostock, Germany); PolyMicrospheres Inc.(Indianapolis, Ind.); Scigen Ltd.(Kent, U.K.); Seradyn Inc.;(Indianapolis, Ind.); and Spherotech Inc. (Libertyville, Ill.). Most ofthese particles are made using conventional techniques, such as grindingand milling, emulsion polymerization, block copolymerization, andmicroemulsion.

[0007] Methods of making silica nanoparticles have also been reported.The processes involve crystallite core aggregation (Philipse et al.,Langmuir, 10:92, 1994); fortification of superparamagnetic polymernanoparticles with intercalated silica (Gruttner, C and J Teller,Journal of Magnetism and Magnetic Materials, 194:8, 1999); andmicrowave-mediated self-assembly (Correa-Duarte et al., Langmuir,14:6430, 1998). Unfortunately, these techniques have not proven to beparticularly efficient for consistently fabricating nanoparticles with aparticular size, shape and size distribution.

SUMMARY OF THE INVENTION

[0008] The invention relates to a new method for preparing nanoparticleshaving a core enveloped by a silica (SiO₂) shell. Such silica-coatednanoparticles can be used, for example, as dye-doped particles,“pigmentless” pigment particles, metal particles, semiconductorparticles, magnetic particles, and drug molecule particles.

[0009] The method employs a microemulsion, i.e., isotropic andthermodynamically stable single-phase system, to produce nanoparticlescores of a predetermined, very uniform size and shape. Cores producedusing the microemuslion are then coated with silica using a silicatingagent. The nanoparticles thus formed can be customized for a particularapplication by derivatizing various chemical groups onto the silicacoating.

[0010] Accordingly, the invention features nanoparticles having a coreand a silica shell enveloping the core. The nanoparticles can have amean size of less than 1 micron (e.g., between 1 nm and 300 nm, orbetween 2 nm and 10 nm). In some variations, the nanoparticle cores canbe magnetic and can include a metal selected from the group consistingof magnetite, maghemite, and greigite. In other variations, the coreincludes a pigment which can be an inorganic salt such as potassiumpermanganate, potassium dichromate, nickel sulfate, cobalt-chloride,iron(III) chloride, or copper nitrate. Similarly, the core can include adye such as Ru/Bpy, Eu/Bpy, or the like; or a metal such as Ag and Cd.

[0011] The invention also features nanoparticles with a silica shellthat is derivatized with a functional group such as a protein (e.g., anantibody); a nucleic acid (e.g., an oligonucleotide); biotin; orstreptavidin.

[0012] Also within the invention is a method of making coatednanoparticles. This method includes the steps of: providing amicroemulsion; providing a first aqueous solution of a first reactantand a second aqueous solution of a second reactant (the first reactantand second reactant being selected such that a solid precipitate formsupon mixing the first and second reactants together in an aqueousenvironment); adding the first aqueous solution to a first aliquot ofthe microemulsion and the second aqueous solution to a second aliquot ofthe microemulsion; mixing together the first and second aliquots to forma reaction mixture that reacts to form nanoparticle cores; and adding acoating agent to the cores to form coated nanoparticles.

[0013] The microemulsion can be a water-in-oil microemulsion that can bemade by mixing together water; a relatively polar liquid such asisooctane, n-hexane, or cyclohexane; a surfactant such as AOT, TX-100,and CTAB; and, in some cases, a cosurfactant such as n-hexanol. Thecoating agent can be a reactive silicate such as TEOS and APTS. In somevariations, the method includes a step of derivatizing the silica shellwith a functional group such as a protein (e.g., an antibody); a nucleicacid (e.g., an oligonucleotide); biotin; or streptavidin. Thus, themethod of the invention can be used to make protein-derivatized,silica-coated nanoparticles having cores including a metal such asmagnetite, maghemite, or greigite.

[0014] In another aspect, the invention features a method of identifyingcells expressing a preselected molecule. This method includes the stepsof: providing a plurality of silica-coated nanoparticles coated with afunctional group that binds to a preselected molecule; providing aplurality of cells at least some of which express the preselectedmolecule; mixing the plurality of silica-coated nanoparticles with theplurality of cells to form a mixture; placing the mixture underconditions that allow the nanoparticles to bind to cells expressing thepreselected molecule; and analyzing the cells for bound nanoparticles.In one variation of this method, the functional group is an antibodythat specifically binds to the preselected molecule. In anothervariation, the silica-coated nanoparticles are fluorescent.

[0015] As used herein, the word “nanoparticle” means a particle having adiameter of between about 1 and 1000 nm. Similarly, by the term“nanoparticles” is meant a plurality of particles having an averagediameter of between about 1 and 1000 nm.

[0016] For the purposes herein, a microemulsion is defined as athermodynamically stable, optically isotropic dispersion of twoimmiscible liquids consisting of nanosize domains of one or both liquidsin the other, stabilized by an interfacial film of surface-activemolecules.

[0017] By reference to the “size” of a nanoparticle is meant the lengthof the largest straight dimension of the nanoparticle. For example, thesize of a perfectly spherical nanoparticle is its diameter.

[0018] By the phrase “specifically binds” means that one moleculerecognizes and adheres to a particular second molecule in a sample, butdoes not substantially recognize or adhere to other molecules in thesample. Generally, an antibody that “specifically binds” a preselectedantigen is one that binds the antigen with a binding affinity greaterthan about 10⁵ to 10⁶ liters/mole.

[0019] As used herein, the phrase “functional group” means a chemicalgroup that imparts a particular function to an article (e.g.,nanoparticle) bearing the chemical group. For example, functional groupscan include substances such as antibodies, oligonucleotides, biotin, orstreptavidin that are known to bind particular molecules; or smallchemical groups such as amines, carboxylates, and the like.

[0020] Unless otherwise defined, all technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions will control. In addition, theparticular embodiments discussed below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention is pointed out with particularity in the appendedclaims. The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

[0022]FIG. 1 is a cross-sectional view of a nanoparticle of theinvention.

[0023]FIG. 2 is a flowchart illustrating general steps involved in amethod of making nanoparticles of the invention.

[0024] FIGS. 3A-D are schematic views illustrating a method of theinvention.

DETAILED DESCRIPTION

[0025] The invention is based on a method for preparing silica-coatednanoparticles using a water-in-oil microemulsion. The method yieldsuniformly-sized particles composed of a core enveloped by a silicashell. The microemulsion is made by combining a relatively polar liquidsuch as water, a relatively non-polar liquid such as a liquid alkane,and one or more surfactants to form an isotropic, thermodynamicallystable single-phase system. This system is comprised of a plurality ofvery small spherical water pools (i.e., reverse micelles) that serve asreactors for producing nanoparticle cores. After the cores are produced,they are coated with silica using a silicating agent such astetraethylorthosilicate (TEOS). In some applications, the silica coatingis derivatized with one or more functional groups to yield nanoparticlesparticularly suited for specific applications. The below describedpreferred embodiments illustrate various adaptations of the invention.Nonetheless, from the description of these embodiments, other aspects ofthe invention can be readily fashioned by making slight adjustments ormodifications to the components discussed below.

[0026] Nanoparticle Characteristics

[0027] In brief overview, referring to FIG. 1, a preferred nanoparticle10 of the invention includes a core 12, a shell 14 coating core 12, andone or more functional groups 16 derivatized onto shell 14. Although thediameter of nanoparticle 10 can range from about 1 nm to about 1000 nmor larger, for many applications it is preferably between about 10 nm toabout 300 nm (e.g., about 10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200,250, or 300 nm). In a dispersion of a plurality of nanoparticles 10, thesize distribution preferably has a standard deviation of no more thanabout 25% (e.g., 1, 2, 3, 5, 10, 15, 20, and 25%) of the averagediameter (or largest straight dimension) of the plurality ofnanoparticles 10.

[0028] The nanoparticle 10 illustrated in FIG. 1 is solid (i.e.,substantially without pores). While this form is preferred for manyapplications, nanoparticles within the invention can also be porous.Solid forms can be prepared as described below by uniformly coating core12 with shell 14. Porous forms can be made by degrading a solidnanoparticle with a corrosive agent (e.g., a very basic solution whereshell 14 is composed of silica), and optionally re-coating core 12 withsilica. In general, solid forms are preferred when it is desired tosequester core 12 from the outside environment; whereas porous forms arepreferred when it is desired to increase the surface area of shell 14 incontact with the outside environment (e.g., where nanoparticle 10 isused a catalyst) or sometimes when nanoparticle 10 is used to isolatevarious substances (e.g., for “trapping” substances within the pores).Pores in nanoparticle 10 can be of any suitable size less than thediameter of nanoparticle 10. For example, such pores can average about0.2, 0.5, 1, 2, 3, 5, 10, 20, 50, or 100 nm in size.

[0029] Core 12 can be composed of any substance compatible with shell14. As core 12 imparts functional characteristics on nanoparticle 10,one skilled in the art can select the composition of core 12 to suit theparticular application intended for nanoparticle 10 based on knowncharacteristics of compositions. For example, in a preferred embodimentwhere nanoparticle 10 is desired to be magnetic, core 12 is made up of amagnetic metal such as magnetite (Fe₃O₄), maghemite (γFe₂O₃), orgreigite (Fe₃S₄). In this example, the composition of core 12 imparts amagnetic quality on nanoparticle 10 such that nanoparticle 10 can beused for magnetically based applications, e.g., cellseparation/purification, diagnostic imaging, recording media, etc.Depending on the particular application, magnetic core 12 can be eithersuperparamagnetic or single-domain (i.e., with a fixed magnetic moment).Superparamagnetic particles are preferred in applications whereparticles having a fixed magnetic moment are not desired; whereassingle-domain particles are preferred when particles having a fixedmagnetic moment are desired, e.g., in magnetic recording media or forbiomedical applications that require mechanical transduction(single-domain particles used to impart a torque).

[0030] For other applications, core 12 can be made up of non-magneticmetals or metal salts (e.g., gold, silver, cadmium sulfide, etc.). Forexample, nanoparticles having CDs cores coated with silica can functioncan be used as quantum dots, i.e., particles having charge carrierssurrounded in all directions by potential barriers and which havequantized energy levels that can be used as highly flourescent orluminescent probes, or semiconductors. As another example, for theproduction of dye or pigment nanoparticles, core 12 can includeinorganic salts useful in preparing “pigmentless” pigments, e.g.,europium salts, tris(2,2′-bipyridyl) dichlororuthenium, potassiumpermanganate, potassium dichromate, nickel sulfate, cobalt chloride,iron(III) chloride, copper nitrate, etc.

[0031] Core 12 can also be composed of a mixture of differentsubstances. For example, where it is desired to make a magnetic,dye-doped nanoparticle, core 12 can be composed of both a magnetic metaland an inorganic salt useful as a pigment. Where core 12 is composed ofa material that is very soluble in common solvents (e.g., thosetypically used in paints and colored-coatings), it is especiallydesirable that such cores be coated with a substance that resistsdissolution or degradation in such solvents.

[0032] Core 12 can be of any size less than the size of nanoparticle 10.Thus, core 12 can have a diameter of between less than 1 and 1000 nm.For many applications, core 12 preferably has a diameter ranging fromabout 1 to about 200 nm. As one example, because animals are able toexcrete nanoparticles sized less than about 100 nm, but retain particlesgreater than 100 nm (primarily in the liver and spleen), cores smallenough to be incorporated in nanoparticles less than 100 nm in size arepreferred in diagnostic or therapeutic applications where is it desiredthat the nanoparticles not be retained in a subject.

[0033] When made using a microemulsion nanoparticle-manufacturingtechnique (see below), core 12 generally has a spheroid shape(conventional reverse micelles are spheroid). Core 12, however, is notlimited to a spheroid shape. For example, rather than being perfectlyround, nanoparticle 10 can be oblong or tube-like, a shape preferred inmany magnetic applications. Where core 12 is in crystalline form,nanoparticle 10 can have a regular or irregular polyhedral shape such asa cuboid shape.

[0034] Shell 14 is a substance that coats core 12. It can be composed ofany compatible material that can be coated onto core 12 using themethods of the invention. Shell 14 can, for example, be composed of apolymer (e.g., polystyrene, polyethylene, polyvinyl chloride, an acrylicpolymer, etc.), a polysaccharide such as dextran, an inorganic oxidesuch as alumina or silica, or mixtures of the foregoing. In thepresently preferred embodiment, shell 14 is composed partially orentirely of silica. Silica is preferred in various applications as it isrelatively inert in many environments, is biocompatible, preventsagglomeration with other nanoparticles in a dispersion, and can bereadily derivatized with many different function groups. And while FIG.1 shows shell 14 configured in a single layer, it can also bemulti-layered. For example, shell 14 can include a first layer of silicacoating and immediately adjacent to core 12, and a second layer coatingthe silica layer. The second layer can be composed of any substance thatbe coated onto the first layer. For example, the second layer can becomposed of a biodegradable material (e.g., a sugar or polymer)impregnated with a drug. When introduced to an animal, the biodegradablematerial and drug will gradually be dissolved into the animal. In otherapplications, shell 14 can be composed of 3, 4, 5 or more separatelayers.

[0035] In the preferred embodiment shown in FIG. 1, shell 14 is showncompletely enveloping core 12 and thus sequestering core 12 from theoutside environment. This form is preferred where it desired to preventinteraction of core 12 with external factors. For example, a silicacoating can prevent corrosion of an iron-based core. Similarly, acomplete silica coating can enhance the shelf life a nanoparticle-basedpigment by preventing degradation or dissolution of the pigment in asolvent or by oxidation. In some variations, nanoparticle 10 does notinclude a shell 14 or is only partially coated with a shell 14 (e.g.,where shell 14 has been partially dissolved or degraded off core 12).

[0036] Shell 14 can be of any thickness (i.e., length from outsidesurface of core 12 to outside surface of shell 14) compatible with themethods of making nanoparticle 10. Using preferred methods of theinvention, shell 14 can be made to have a thickness ranging from lessthan about 1 nm to greater than about 300 nm. Depending on theparticular application that nanoparticle 10 is to be used in, thepreferred thicknesses of shell 14 will vary. For example, a relativelythick shell is generally preferred where it is desired to reduceagglomeration of nanoparticles (where the cores attract one another) ordegradation of the shell (e.g., in a caustic solvent). On the otherhand, where it is desired to amplify the properties of the core (e.g.,color of a pigment), a relatively thinner shell is generally preferred.

[0037] As shown in FIG.1, functional groups 16 can be derivatized ontothe surface of shell 14. Functional groups 16 can take the form of anychemical or biological group that can be attached to nanoparticle 10 viashell 14. For example, functional groups 16 can be one or more ofproteins such as antibodies (monoclonal, polyclonal), enzymes, biotin,and streptavidin; nucleic acid molecules (e.g., RNA, DNA); chemosensorssuch as fluorescent probes; and biochemical groups such as amines andcarboxylates.

[0038] Methods of Making Nanoparticles

[0039] Referring now to FIG. 2, a preferred method of makingnanoparticles includes: a step 50 of providing a microemulsion; a step52 of providing aqueous solutions of reactants; a step 54 of adding theaqueous solutions to separate aliquots of the microemulsion; a step 56of mixing the aliquots to form a reaction mixture that producesnanoparticle cores; and a step 58 of adding a coating agent to the coresto form coated nanoparticles.

[0040] The microemulsion of step 50 can be made by mixing together atleast two immiscible liquids in the presence of at least one surfactantto form a thermodynamically stable, optically isotropic dispersion ofnanosize droplets of one or both liquids in the other. The dispersion isstabilized by the surfactant reducing the surface tension at theinterface of the two liquids. Microemulsions can be either water-in-oil(i.e., reverse micelles or water droplets dispersed in oil),oil-in-water (i.e., micelles or oil droplets dispersed in water), or abi-continuous system containing comparable amounts of two immisciblefluids. In some cases, microemulsions can be made by mixing together twonon-aqueous liquids of differing polarity with negligible mutualsolubility. For use in the invention water-in-oil microemulsion arepresently preferred because they are compatible with very many knownchemical reactions for precipitating solids in aqueous solutions.

[0041] The immiscible liquids that can be used in step 50 typicallyinclude a relatively polar (i.e., hydrophobic) liquid and a relativenon-polar (i.e., hydrophillic) liquid. While a large variety ofpolar/nonpolar liquid mixtures can be used to form a microemulsionuseful in the invention, the choice of particular liquids utilized willdepend on the type of nanoparticles being made. A skilled artisan canselect specific liquids for particular applications by adapting knownmethods of making microemulsions for use in the present invention. Thepresently preferred relatively polar liquid is water, although otherpolar liquids might also be useful. Water is preferred because it isinexpensive, readily available, non-toxic, easy to handle and store,compatible with a large number of different precipitation reactions, andimmiscible in a large number of nonpolar solvents. Examples of suitalenon-polar liquids include alkanes (e.g., any liquid form of hexane,heptane, octane, nonane, decane, undecane, dodecane, etc.), cycloalkanes(e.g., cyclopentane, cyclohexane, etc.), aromatic hydrocarbons (e.g.,benzene, toluene, etc.), and mixtures of the foregoing (e.g., petroleumand petroleum derivatives). In general, any such non-polar liquid can beused as long as it is compatible with the other components used to formthe microemulsion and does not interfere with the involved precipitationreaction.

[0042] Step 50 requires at least one surfactant to form a microemulsion.Surfactants are surface active agents that thermodynamically stabilizethe very small dispersed micelles or reverse micelles in microemulsions.Typically, surfactants possess an amphipathic structure that allows themto form films with very low interfacial tension between the oily andaqueous phases. Thus, any substance that reduces surface tension at theinterface of the relatively polar and relatively non-polar liquids andis compatible with other aspects of the invention can be used to formthe microemulsion used to make nanoparticles. The choice of a surfactantwill depend on the particular liquids utilized and on the type ofnanoparticles being made. Specific surfactants suitable for particularapplications can be selected from known methods of making microemulsionsor known characteristics of surfactants. For example, non-ionicsurfactants are generally preferred when an ionic reactant is used inthe microemulsion process and an ionic detergent would bind to orotherwise interfere with the ionic reactant.

[0043] Numerous suitable surfactants are known. A nonexhaustive listincludes soaps such as potassium oleate, sodium oleate, etc.; anionicdetergents such as Aerosol® OT, sodium cholate, sodium caprylate, etc.;cationic detergents such as cetylpyridynium chloride,alkyltrimethylammonium bromides, benzalkonium chloride,cetyldimethylethylammonium bromide, etc; zwitterionic detergents such asN-alkyl-N,N-dimethylammonio-1-propanesulfonates and CHAPS; and non-ionicdetergents such as polyoxyethylene esters, polyoxyethylene ethers,polyoxyethylenesorbitan esters, sorbitan esters, and various tritons(e.g., (TX-100, TX-114); etc.

[0044] The concentration of surfactant used in step 50 will depend onmany factors including the particular surfactant selected, liquids used,and the type of nanoparticles to be made. Suitable concentrations can bedetermined empirically, i.e., by trying different concentrations ofsurfactant until the concentration that performs best in a particularapplication is found. Ranges of suitable concentrations can also bedetermined from known critical micelle concentrations.

[0045] In preferred embodiments bis (2-ethylhexyl) sulfosuccinate sodiumsalt (Aerosol® OT, AOT) is used to create a microemulsion of water andisooctane; cetyltrimethylamnmonium bromide (CTAB) is used to create amicroemulsion of n-hexane, n-hexanol, and water; and triton X-100(TX-100) is used to make a microemulsion of cyclohexane, n-hexanol, andwater.

[0046] Although, in most applications the invention, step 50 employsonly one surfactant to stabilize the microemulsion, one or morecosurfactants can also be used. The use of a cosurfactant is sometimesadvantageous for stabilizing reverse micelle systems. For example,adding an aqueous surfactant such as soap to a mixture of oil and wateryields a milky emulsification. Adding a co-surfactant such as an alcoholof intermediate chain length causes the milky emulsion to clearspontaneously due to formation of very small spheres of dispersed waterdroplets in oil. Such cosurfactants function by further reducing theinterfacial tension between the phases to facilitate the formation ofvery small particles of dispersed phase. Suitable cosurfactants for usein the invention include hexanol, butanol, pentanol, octanol, and likeintermediate chain length alcohols.

[0047] The microemulsion of step 50 is prepared by simply mixingtogether a relatively polar liquid, a relatively non-polar liquid, andone or more surfactants. For preparing a water-in-oil microemulsion(having aqueous reverse micelles), the volume of the relativelynon-polar liquid vastly exceeds that of the relatively polar liquid(e.g., non-polar liquid:polar liquid volume ratio between about 10000:1to 100:1). While addition of the surfactant can sometimes cause amicroemulsion to form without further agitation, generally the mixtureis mechanically (e.g., magnetically) stirred or ultrasonicated to formthe microemulsion. Many microemulsions useful in the invention can beprepared at room temperature (i.e., about 20° C.) without addition ofheat. In other cases, to hasten microemulsion formation by increasingthe solubility of the surfactant in the liquids, the mixture ofingredients is sometimes heated (e.g., using a hot plate) to betweenabout 25-80° C.

[0048] Referring again to FIG. 2, step 52 of providing aqueous solutionsof reactants and step 54 of adding the aqueous solutions of step 52 toseparate aliquots of a microemulsion can be performed using awater-in-oil microemulsion prepared as described above. Steps 52 and 54can be accomplished by first providing a first water-soluble reactant(reactant A) and a second water-soluble reactant (reactant B), and thenadding reactant A to a first aliquot of a water-in-oil microemulsion andreactant B to a second aliquot of a water-in-oil microemulsion. The twoaliquots are separately mixed until reactant A reaches equilibriumdistribution in each reverse micelle (reverse micelles continuouslyform, coalesce, and break apart in the microemulsion, thereby allowingany reactant contained therein to be distributed equally among thereverse micelles) of the first aliquot, and reactant B reachesequilibrium distribution in each reverse micelle of the first aliquot.In step 56, after allowing for the distribution of the dissolved speciesto equilibrate, the two aliquots are mixed together. Due to collisionand coalescence of the reverse micelles, the cations of reactant A andanions of reactant B contact each other and react to form precipitatesthat serve as nanoparticle cores.

[0049] Reactants A and B are generally selected so that they can reactto form a precipitate within the reverse micelles of the microemulsions.They are typically soluble in the aqueous reverse micelles and may besolids, liquids, or gases. In a preferred embodiment, Reactant A is asalt (e.g., with the hypothetical formula A⁺X⁻) that dissolves intosoluble cations (e.g., A⁺'s) within the reverse micelles of the firstaliquot of the microemulsion, and Reactant B is another salt (e.g., withthe hypothetical formula B⁺Y⁻) that dissolves into soluble anions (e.g.,Y⁻'s) within the reverse micelles of the second aliquot of themicroemulsion. The cations of Reactant A and anions of Reactant B areselected so that they form a precipitate (A⁺Y⁻)when mixed together in anaqueous solution.

[0050] While the foregoing illustrates a preferred method of theinvention, other methods for making nanoparticle cores usingmicroemulsions are also within the invention. Many of these can beperformed by making slight modifications to the preferred method justdescribed. For example, rather than mixing together two differentaliquots of a microemulsion, the core-forming reaction can be carriedout using a single aliquot of a microemulsion. In this case, a reactantcan be added to the single aliquot and allowed to dissolve andequilibrate among the reverse micelles of the microemulsion.Subsequently, a precipitating (e.g., reducing or oxidizing) agent in theform of a liquid or gas (e.g., hydrogen, hydrazine, NH₄OH) is added tothe single aliquot to precipitate the reactant dissolved in the reversemicelles.

[0051] Nanoparticle cores can be isolated from a microemulsion by addinga solvent such as acetone or ethanol to the microemulsion and thenfiltering and/or centrifuging the mixture to isolate the nanoparticles.For filtering, filters have pores sized smaller than the nanoparticles.For centri-fuging, the mixture can be spun at 10,000 RPM or more in amicrocentrifuge for 15 minutes or more to pellet the nanoparticles andthe supernatant can be decanted. Nanoparticles isolated in this mannercan be washed one or more times with acetone or an ethanol/watersolution to remove any surfactant or other microemulsion component. Theisolated and washed nanoparticles can be dried over acetone. Prior touse or functionalization, the nanoparticles can be resuspended in anappropriate liquid.

[0052] Using the water-in-oil microemulsion technique, nanoparticle coresize is highly controllable. Although core size generally relates toreverse micelle size, this is not necessarily a strict relationship ascore size does not always correlate with the amount of reactant(s)originally present in each reverse micelle. For example, even smallnanoparticle cores (e.g., having diameters of 2 nm to 5 nm) contain fromabout 300 to 1000 atoms, which is in most cases appreciably larger thanthe number of reactant molecules present in each micelle prior toreaction. This indicates that nanoparticle core nuclei first form in asmall fraction of micelles; these then consume the reactant(s) in othermicelles through collision-coalescence processes.

[0053] A factor to consider in nanoparticle core preparation thereforeis the rate at which nanoparticle cores form. The rate at whichnanoparticle cores form directly relates to the rate at which thereverse micelles coalesce. Thus, the specific surfactant selectedstrongly influences the core formation rate, controlling the rate ofreverse micelle coalescence. That is, surfactants that result in arelatively rigid interface between the two immiscible liquids of themicroemulsion decrease the core formation rate, while surfactants thatresult in a fluid interface increase the rate. Other properties of themicroemulsion, such as ionic strength, pH, and temperature can also bemanipulated to control the rate of core formation.

[0054] Through empirical adjustment of initial reactant concentrationsand microemulsion compositional parameters, nanoparticle cores withhomogeneous size distribution (e.g., percentage standard deviation incore size is between about 1 and 5% (for instance, 1, 2, 3, 4, and 5%))and average diameters ranging from about 1 nm to about 300 nm or more.Cores of larger size (e.g., about 1 micron) can be prepared by: (i)adding a higher concentration of reagent(s) to the reaction medium(e.g., reverse micelles of the microemulsion), and/or (ii)sonochemically (i.e., by ultrasonication) dispersing isolated cores in asuitable solvent other than microemulsion to make a uniform coresuspension, and then adding additional reagent to the dispersion. In thelatter method, individual cores often fuse.

[0055] In most cases, nanoparticle cores made according to thewater-in-oil microemulsion technique described above have a spheroidshape (conventional reverse micelles are spheroid). By altering variousparameters in the core formation process, it is possible to producecores having other shapes. For example, oblong or tube-shaped cores canbe made by adding very high concentration of sodium dodecyl sulfate tothe microemulsion. As another example, where reactants are selected suchthat the formed cores have a crystalline structure, nanoparticle coreshaving a regular or irregular polyhedral shape can be made.

[0056] Magnetic nanoparticles can be made using magnetic materials suchas magnetite, maghemite, and greigite as part of the core. By varyingthe overall size and shape of such magnetic cores, they can be madesuperparamagnetic or stable single-domain (particles that retain astable magnetic moment after being removed from a magnetic field). Coresize relates to whether a magnetic nanoparticle is superparamagnetic orsingle-domain. Thus, relatively equidimensional superparamagneticparticles generally have a core sized less than 50 to 80 nm. At particlesizes above this upper range, the magnetization of the particle is splitinto domains of differing magnetization vectors in order to minimizeinternal magnetic energies.

[0057] Referring once again to FIG. 2, methods of making nanoparticleswithin the invention feature a step 58 of adding a coating agent to formcoated nanoparticles. The coating agent used in step 58 can be any thatcauses silica (or another substance) to be deposited onto the surface ofthe nanoparticle cores. Presently preferred reagents include reactivesilicates such as tetraethylorthosilicate (TEOS) oraminopropyltrimethoxysilane (APTS) (both available from Sigma, St.Louis). To coat cores, such reactive silicates are simply added to asolution of nanoparticle cores (e.g., the microemulsion in which thecores were prepared) along with a reducing agent such as ammoniumhydroxide or NaOH. The mixture can be stirred for a suitable amount oftime to allow the cores to become coated with silica.

[0058] Thickness of the silica coating, and the reaction rate for theformation of silica coating are dependent on the amount of reactivesilicate added, reaction time, amount of reducing agent added, andreverse micelle size (where coating is performed in a water-in-oilmicroemulsion). Increasing the concentration of the reducing agent (e.g,[NH₄OH]) to reactive silicate concentration (e.g., [TEOS]) generallyresults in a thicker coating forming after a given reaction time.Increasing the concentration of polar liquid (e.g., water) to reactivesilicate concentration generally results in a thinner coating formingafter a given reaction time. The precise reaction conditions forcontrolling the thickness of the coating will vary according to theparticular agent used, the core material, etc. These, however, can bedetermined empirically by simple experiments varying the concentrationsof reagents and reaction times and conditions.

[0059] Methods within the invention can also include a step offunctionalizing (i.e., derivatizing with one or more functional chemicalgroups) coated nanoparticles made as described above. Numerous knownmethods for attaching functional groups to silica can be adapted for usein the present invention. See, e.g., Ralph K. Iler, The Chemistry ofSilica: Solubility, Polymerization, Colloid and Surface Properties andBiochemistry, Wiley-Interscience, NY, 1979; VanDerVoort, P. and Vansant,E. F., Journal of Liquid Chromatography and Related Technologies,19:2723-2752, 1996; and Immobilized Enzymes Antigens Antibodies, andPeptides: Preparation and Characterization, Howard H. Weetall (ed.), M.Dekker, NY, 1975. A typical process for adding functional groups tosilica-coated nanoparticles involves treating the nanoparticles with asilanizing agent that reacts with and couples a chemical group to thesilica surface of the nanoparticles. The chemical group can itself bethe functional group, or it can serve as a substrate to which functionalgroups can be coupled.

[0060] For example, in an exemplary method, silica-coated nanoparticlesare prepared as described above and the particle surfaces are silanizedusing trimethylsilylpropyl-diethylenetriamine (DETA), a silanizationagent that attaches primary amine groups to silica surfaces. Antibodiesor other proteins can then be covalently coupled to the silanizedsurface using the cyanogen bromide (CNBR) method. As one example,CNBR-mediated coupling can be achieved by suspending silica-coatednanoparticles previously silanized with DETA in a 2 M sodium carbonatebuffer and ultrasonicating the mixture to create a particle suspension.A solution of CNBR (e.g., 2 g CNBR/1 ml acetonitirile) is then added tothe particle suspension to activate the nanoparticles. After washing thenanoparticles with a neutral buffer (e.g., PBS, pH 8), an antibodysolution is added to the activated nanoparticle suspension causing theantibodies to become bound to the nanoparticles. A glycine solution canalso be added to the antibody-coated nanoparticles to block anyremaining unreacted sites.

[0061] Methods of Using Nanoparticles

[0062] Nanoparticles of the invention to isolate cells (e.g., eukaryoticor prokaryotic cells). One such method is illustrated in FIG. 3.Referring to FIG. 3A, antibody-derivatized magnetic nanoparticles 10 areshown mixed with target cells 20 and non-target cells 21 in container30. Target cells 20 express a target antigen 22 on their surface, whilenon-target cells 21 do not. In the nanoparticles shown, core 12 includesa ferrous material such as magnetite or maghemite, and functional groups16 include an antibody that can specifically bind target antigen 22.Referring now to FIG. 3B, nanoparticle 10 is shown physically bindingtarget cell 20 via the interaction of functional groups 16 and targetantigen 22. Such binding spontaneously results when nanoparticle 10 andtarget cell 20 are mixed together in container 30 under conditions whichallow antibody-antigen binding (e.g., about room temperature, neutral toslightly basic pH in a low salt buffer). Non-target cells 21 do notspecifically bind nanoparticles 10 because they do not express targetantigen 22. As shown in FIG. 3C, application of a magnetic field to thenanoparticle-cell mixture by insertion of magnet 32 into container 30causes nanoparticles 10 and bound target cells 21 to associate withmagnet 32. Referring to FIG. 3D, by removing magnet 32 from container30, target cells 20 can be isolated. Cells 20 can be separated fromnanoparticles using an excess of soluble antigen.

[0063] Many other applications are specifically envisioned including,for example, cell labeling (see Example 8 below), targeted drug or genedelivery, biosensors, magnetic recording media, magnetic resonanceimaging, and use in micro- or nano-sized machines. For example,cytotoxic drugs or viral vectors carrying therapeutic genes can beattached to the functional groups on the surface of nanoparticles. Thesenanoparticles can then be dispersed in a pharmaceutically acceptablecarrier (e.g., USP grade saline) and administered to a patient (e.g., byintravenous injection). Magnetic fields can then be used to concentratethe virus or drug at the delivery site to enhance site-specific uptake(e.g., by placing a magnet at the site). Drugs coated onto nanoparticlescan be further contained within a time-release coating (e.g., abiodegradable sugar) so that the drug can accumulate at the site beforebecoming active.

[0064] In other envisioned examples, fluorescence-based biosensors canbe attached to the particles. The resulting particles can be manipulatedby magnetic means into specific target sites (specific locations inisolated cells), and used to monitor biochemical processes in situ. Thenanoparticles of the invention are also thought to be useful forenhancing Magnetic Resonance Images (MRI). For example, as describedabove, antibody or ligand-coated nanospheres can be caused to accumulateat sites in the body where the target antigen or receptor isconcentrated or located. In comparison to non-targeted MRI contrastagents, the increased concentration of particles at a targeted site willenhance the contrast in an MRI.

[0065] Nanoparticles manufactured in a stable, single domain size rangethat allows a remnant magnetization to be preserved are envisioned to beuseful in binary magnetic recording applications where they can besubstituted for the simple iron particles used in conventional magneticstorage devices. For example, arrays of ferrite-doped silica particlescould be tailored for minimum magnetostatic interactions to permitindividual nanoparticles to be magnetized either parallel orantiparallel to their easy axis of magnetization for binary data storageapplications. And because stable single domain particles are able totransduce applied magnetic fields as mechanic motion (i.e. a torque canbe exerted on the particle when a magnetic field is applied at an angleto the easy axis of magnetization), arrays of these nanoparticles of theinvention could find use as mechanical micro- or nanomachines. Oneparticular example would be micromechanical gate activation uponapplication of an external magnetic field.

EXAMPLES Example 1 Preparation of Silica-Coated Magnetite Nanoparticlesby Microemulsion

[0066] (A) A 0.27 M bis (2-ethylhexyl) sulfosuccinate sodium salt(Aerosol OT or AOT) solution was prepared by dissolving 12 g AOT in 10ml isooctane. An aliquot of ultra-pure water was purged for one hourwith N₂ gas. A stock solution of 1 M Fe(II) was prepared by dissolving0.278 g FeSO₄7H₂O in 1 ml of the nitrogen purged water. Similarly, astock solution of 1.5 M Fe(III) was prepared by dissolving 0.4055 gFeCl₃6H₂O (0.4055 gm) in 1 ml of the nitrogen purged water. In a glasscontainer, 25 μl of the 1 M Fe(II) solution and 25 μl of the 1.5MFe(III) solution were added to a 5 ml aliquot of the AOT solution undera nitrogen atmosphere, and the resulting Fe/AOT mixture was magneticallystirred for 1 hr to form a Fe/AOT solution. In another container, 100 μlNH₄OH (28-30 wt %) was added to another 5 ml aliquot of the AOTsolution, and the resulting NH₄OH/AOT mixture was magnetically stirredfor 1 hr to form a NH₄OH/AOT solution. In absence of magnetic field, theNH₄OH/AOT solution was added dropwise to the Fe/AOT solution withvigorous mechanical stirring for 1 hr. Initially a light yellow solutionformed. This solution turned brown (without any precipitate formation)as magnetite nanoparticles formed. 50 μl of tetraethylorthosilicate(TEOS) was then added to the resulting brown solution and mechanicalstirring was continued for an additional 24 hrs. Silica-coateduniform-sized nanoparticles in powder form were obtained by coagulatingthe colloidal microemulsion with acetone, and then filtering and washingthe particles with acetone and ethanol several times with each solvent.In some cases, 50% (v/v) solution of ethanol/water was also used forwashing.

[0067] (B) 1.78 g of AOT was dissolved in 20.0 ml of isooctane. Theresulting solution was mixed using a sonicator and flushed withnitrogen. 100 μL each of N₂-flushed 0.10 M FeSO₄ and 0.15 M FeCl₃ (bothprepared in water) were then added and mixed into the solution to form amicroemulsion. 100 μL of N₂-flushed solution of 2.0 M NaOH was thenadded, and the resulting microemulsion was sonicated for 1 hour whilebeing continuously flushed with N₂. 10 μL of TEOS was then added to thesonicated microemulsion, and the mixture was allowed to react overnight.The isooctane contained in the microemulsion was then evaporated and theremaining gel was dissolved in ethanol. This solution was centrifuged topellet the nanoparticles and the supernatant was discarded. Afterwashing 3 or 4 more times with ethanol, the resulting nanoparticles weresubjected to TEM which showed that nanoparticles having a diameter ofapproximately 3-5 nm were produced. A silica shell was observed as atranslucent halo about 2-3 nm thick surrounding the denser core.

[0068] (C) A microemulsion I (ME I) was prepared by first dissolving8.89 g AOT in 40.0 ml isooctane to form a first solution. 1.2 ml H₂O and6.0 ml FeSO₄ was then added and the mixture was sonicated to form ME I.In another glassware, a microemulsion II (ME II) was similarly preparedby first dissolving 8.89 g AOT and dissolve in 40.0 ml isooctane to forma second solution. 3.2 ml H₂O and 4.0 ml NH₄OH was added to the secondsolution, which was then mixed for about 30 minutes to form ME II. 10 μLTEOS was added to ME II, and the mixture was allowed to react for about2 hours. Using a glass syringe, ME II was slowly added to ME I, and thereaction mixture thus formed was sonicated for 24 hours. As described in(B) above, the isooctane was then evaporated, the remaining gel wasdissolved in ethanol and centrifuged to recover the formed nanoparticleswhich were washed 3 or 4 times with additional ethanol. TEM showed thatnanoparticles having a diameter of approximately 25 nm were produced. Asilica shell was observed as a translucent halo about 5 nm thicksurrounding the denser core.

Example 2 Preparation of Dye-doped Silica-Coated Nanoparticles

[0069] (A) Preparation of Eu/Bpy (Eu³⁺/2,2′-dipyridyl)-doped silicananoparticles in cetyltrimethylammonium bromide(CTAB)/n-hexane/n-hexanol (cosurfactant)/water water-in-oilmicroemulsions. 90 ml of a water-in-oil microemulsion stock solution wasprepared by mixing together 2.916 g CTAB, 75 ml n-hexane, 15 mln-hexanol and 880 μl water using a magnetic stirrer. 10 ml of the stocksolution was equally divided into two 5 ml aliquots. 50 μl T'EOS and 5μl 0.1 M Eu/Bpy (aqueous solution) was added to one of the 5 ml aliquotsand the mixture was stirred for 1 hr to form a TEOS/Eu/Bpy solution. 137μl NH₄OH was added to the other 5 ml aliquot and the mixture was stirredfor 1 hr to form an NH₄OH solution. The NH₄OH solution was then addeddropwise to the TEOS/Eu/Bpy solution and the resulting mixed solutionwas stirred overnight. The water to surfactant molar ratio of the mixedsolution was 15 (water:surfactant). Eu/Bpy-doped silica nanoparticleswere isolated in powder form by adding 25 ml of acetone to themicroemulsion of the mixed solution, centrifuging the resultant mixturefor 15 minutes at 10,00 RPM in a microcentrifuge to pellet thenanoparticles, the supernatant was removed and the remainingnanoparticles were washed several times with acetone or an ethanol/watersolution to further remove surfactant and other microemulsioncomponents. The washed nanoparticles were then dried over acetone.

[0070] (B) Ru/Bpy [Ru^(II)(Bpy)₃]-doped silica nanoparticles in tritonX-100 (TX-100)/cyclohexane/n-hexanol(cosurfactant)/water water-in-oilmicroemulsions. 10 ml of a water-in-oil microemulsion was prepared bymixing 7.5 ml cyclohexane, 1.8 ml n-hexanol, 1.77 ml TX-100, 340 μlwater and 140 μl 0.1 M Ru^(II)(Bpy)₃ (aqueous solution) for 1 hr with amagnetic stirrer. The resulting solution was then divided into two 5 mlaliquots. 100 μl TEOS was added to one aliquot and the mixture wasstirred for 30 minutes to form a TEOS solution. 60 μl of NH₄OH was addedto the other 5 ml aliquot and the mixture was stirred for 30 minutes toform a NH₄OH solution. The NH₄OH solution was then added to the TEOSsolution dropwise for a period of 10 minutes and the resulting mixedsolution was stirred overnight. Ru/Bpy doped silica nanoparticles wereisolated as described above in (A).

Example 3 Preparation of Metal-Doped Silica-Coated Nanoparticles

[0071] 10 ml of aTX-100/cyclohexane/n-hexanol(cosurfactant)/waterwater-in-oilmicroemulsion stock solution was prepared as described in Example 2(A).The stock solution was equally divided into two 5 ml aliquots. 30 μl ofa 1M aqueous solution of silver nitrate (AgNO₃) was added to one of the5 ml aliquots and the mixture was stirred for about 30 minutes to form aAgNO₃ solution. 11 μl of a 2M aqueous solution of sodium borohydride(NaBH₄) was added to the other 5 ml aliquot and the mixture was stirredfor about 30 minutes to form an NaBH₄ solution. The NaBH₄ solution wasthen added dropwise to the AgNO₃ solution for the period of 15 minutesto form a reaction mixture. After 5 minutes, 10 μl of TEOS was added andthe resulting mixture stirred for another 15 minutes. 10 μl of a NH₄OHsolution was then added and stirring was continued overnight. TheAg-doped silica nanoparticles were isolated similar to the proceduredescribed in Example 2(A), i.e., by adding 25 ml of acetone to themicroemulsion, filtering, washing several times with an ethanol/watersolution to remove surfactant, and finally drying over acetone.

[0072] Using a variation of this method cadmium sulfide (CDs)-dopedsilica nanoparticles were also prepared. In this case, cadmium nitrateand ammonium sulfide were used in place of silver nitrate and sodiumborohydride, respectively.

Example 4 Preparation of Pigments

[0073] A new class of pigments was prepared using regular inorganicsalts including potassium permanganate, potassium dichromate, nickelsulfate, cobalt-chloride, iron(III) chloride, and copper nitrate. Whilethese salts are highly water soluble, they become completely insolubletrip when coated with silica, and thus behaved as pigments. All werecolored and photostable.

[0074] These pigments was prepared as described for the Ru/Bpy dopedsilica nanoparticles described above in Example 2(B), except that a 0.1Msalt solution (e.g., potassium permanganate, potassium dichromate,nickel sulfate, cobalt-chloride, iron(III) chloride, and copper nitrate)was used in place of the 0.1 M Ru^(II)(Bpy)₃ solution, and an additional100 μl TEOS and 60 μl NH₄OH were added to the mixed solution 12 hrsafter the 10 ul addition of NH₄OH. Pigment particles were separated fromthe mixture after 24 hrs.

Example 5 Characterization of Nanoparticles

[0075] Transmission electron microscopy (TEM) and other analyses wereused to characterize of the size of various nanoparticles made accordingto the invention. As one example, Ru^(II)(Bpy)₃ nanoparticles wereprepared as described in Example 2(B) and subjected to TEM. By analyzingphotographs of the TEM images, it was determined that the Ru^(II)(Bpy)₃nanoparticles had a core size of about 20 nm (standard deviation=+/−2nm) and an overall particle size of about 100 nm (standarddeviation=+/−10 nm). Smaller and larger silica-coated nanoparticles werealso prepared by varying the preparation conditions specified above. Forexample, those with core sizes even as small as 2 nm (e.g., sized toprepare quantum dots) with constant or varied thickness (as large as 300nm) of the outer silica coating have been prepared. Nanoparticles usefulas pigments (see Example 5) were also subjected to TEM which showed thatsuch particles were sized between 0.2 and 0.3 μm. In other analyses,compared to commercially available conventional dyes, dye-dopednanoparticles made according to the invention proved extremely resistantto bleaching even after strong excitation from a laser source.Similarly, no fluorescence quenching was observed in fluorescentdye-doped nanoparticles.

Example 6 Preparation of a Nanoparticle-based Chemosensor

[0076] Dye-doped, silica-coated nanoparticles were prepared using awater-in-oil microemulsion technique. The water-in-oil microemulsion wasprepared first by mixing Triton X-100 (TX-100), cyclohexane, n-hexanol(4.2:1:1 VN) and an adequate amount of water. An aqueous dye solution(Ru^(II)(Bpy)₃; (see example 3B above) was then added to themicroemulsion in such a way that the water to surfactant molar ratio waskept constant at 10. The final dye concentration in the mixture was 0.1M. TEOS was then added to the mixture. A polymerization reaction wasinitiated by adding NH₄OH (volume ratio of TEOS to NH₄OH was 1.7), andthe reaction was allowed to continue for 12 hours. After the reactionwas complete, the dye-doped silica nanoparticles were isolated by addingacetone to the reaction, followed by centrifuging and washing withethanol and water for several times. The nanoparticles were then storedin aqueous solution for later usage.

[0077] The dye-doped silica nanoparticles produced were uniform in size,as characterized by transmission electron microscopy (TEM) and atomicforce microscopy (AFM). A TEM image of the dye-doped silica particlesshowed that the particles were 60±10 nm in size and uniform. At a higherresolution, the luminescent complex of RuBpy dye aggregates were alsovisible as darker dots embedded inside the silica sphere due to thepresence of heavy metal atom in these dye molecules. These individualdye aggregates were as small as 1 nm.

[0078] To investigate whether the RuBpy molecules doped inside thesilica network could function as an oxygen sensor, the fluorescenceemission spectra of free RuBpy molecules was compared to that of theRuBpy-doped nanoparticles at various air pressures. With the free RuBpydye molecules, the intensity of the emission was greatly decreased asthe air pressure was increased from 1 to 14 psi. In contrast, under thesame conditions, no significant change in the emission spectra for thedye-doped silica-coated nanoparticles was observed, indicating that thesilica network was essentially impermeable to oxygen molecules. In otherexperiments, the dye-doped silica-coated nanoparticles also showedexcellent photostability even upon intensive laser illumination.

Example 7 Functionalized Silica-Coated Nanoparticles

[0079] The dye-doped, silica-coated nanoparticles of Example 6 werederivatized with antibodies by first silanizing the particle surfaceswith DETA, a silanization agent that attaches the primary amine group tosilica surfaces. Using fluorescamine, a non-fluorescent molecule thatbecomes highly fluorescent upon reacting with the primary aliphaticamine group (Cordek, J. Wang, X and Tan W., Anal Chem, 71, 1529-1533,1999; Chung, L. A. Anal Biochem. 1997,248,195), the presence of aminegroup on the surface of the nanoparticles was confirmed.

[0080] After surface silanization with DETA, an antibody (mouseanti-human CD10) was immobilized onto the silanized silica surface usingthe cyanogen bromide (CNBR) method. Dye-doped particles (26 g) wereprepared as described in Example 6, dried, and then suspended in 9.0 ml2 M sodium carbonate solution (activation buffer) using ultrasonication.A solution of CNBR in acetonitrile (1.0 gm of CNBR dissolved in 0.5 mlacetonitrile) was then added dropwise to the particle suspension (10mg/ml) under stirring for 5 minutes at room temperature. The resultingCNBR-activated particles were washed twice with ice-cold water and twicewith PBS buffer (pH 8.0).

[0081] 40 μl of the antibody diluted in PBS buffer (pH 8.0) was thenadded to the surface modified particles, and stirring was continued for24 hours at 4° C. The resulting antibody-derivatized nanoparticles werethen treated with 10 ml of 0.03 M glycine solution for 30 minutes toblock any remaining reactive sites. The final product was washed,re-suspended in PBS (pH 8.0) buffer and stored at 4° C. for futureusage. No change in the optical and spectroscopic properties of thenanoparticles was observed.

Example 8 Cell Labeling

[0082] Mononuclear lymphoid cells (about 2 million cells/ml) wereobtained as a suspension in the cell culture medium. The cell suspensionwas incubated for 2 hours with the anti-CD10 immobilized nanoparticlesdescribed in Example 7. After incubation, the cell suspension was imagedwith both optical microscopy and fluorescence microscopy. Themicroscopic analysis revealed that most of the cells were labeled(indicated by the bright emission of the dye-doped particles). Theoptical images correlated well with the fluorescence images. In controlexperiments using non-antibody derivatized dye-doped nanoparticles, nolabeling of cells was observed. In the labeled cells, thesignal-to-noise ratio (i.e., the ratio between the intensities of thebright and the dark areas in the fluorescence image) was over 500.

OTHER EMBODIMENTS

[0083] While the above specification contains many specifics, theseshould not be construed as limitations on the scope of the invention,but rather as examples of preferred embodiments thereof. Many othervariations are possible. Accordingly, the scope of the invention shouldbe determined not by the embodiments illustrated, but by the appendedclaims and their legal equivalents.

What is claimed is:
 1. A method of identifying cells expressing apreselected molecule comprising the steps of: providing a plurality ofcells at least some of which express the preselected molecule; providinga plurality of silica-coated nanoparticles coated with a functionalgroup that binds to the preselected molecule, each of said nanoparticlescomprising a core and a silica shell; mixing the plurality ofsilica-coated nanoparticles with the plurality of cells to form amixture; placing the mixture under conditions that allow thenanoparticles to bind to cells expressing the preselected molecule; andanalyzing the cells for bound nanoparticles.
 2. The method of claim 1,wherein silica-coated nanoparticles are fluorescent.
 3. The method ofclaim 1, wherein the plurality of nanoparticles have a mean size of lessthan 1 micron.
 4. The method of claim 1, wherein the nanoparticles havea mean size between 1 nm and 300 nm.
 5. The method of claim 1, whereinthe nanoparticles have a mean size between 2 nm and 10 nm.
 6. The methodof claim 1, wherein the core is magnetic.
 7. The method of claim 6,wherein the core comprises a metal selected from the group consisting ofmagnetite, maghemite, and greigite.
 8. The method of claim 1, whereinthe core comprises a pigment.
 9. The method of claim 8, wherein thepigment is an inorganic salt selected from the group consisting of:potassium permanganate, potassium dichromate, nickel sulfate,cobalt-chloride, iron(III) chloride, and copper nitrate.
 10. The methodof claim 1, wherein the core comprises a dye selected from the groupconsisting of Ru/Bpy and Eu/Bpy.
 11. The method of claim 1, wherein thecore comprises a metal selected from the group consisting of Ag and Cd.12. The method of claim 1, wherein the functional group is a protein.13. The method of claim 12, wherein the functional group is an antibodythat specifically binds to the preselected molecule.
 14. The method ofclaim 13, wherein the core comprises a metal selected from the groupconsisting of magnetite, maghemite, and greigite.
 15. The method ofclaim 1, wherein the functional group is a nucleic acid.
 16. The methodof claim 1, wherein the functional group is a substance selected fromthe group consisting of biotin and streptavidin.
 17. The method of claim1, wherein the silica shell comprises a reactive silicate selected fromthe group consisting of TEOS and APTS.